Method for treating a degenerative neurological disorders comprising administering asm inhibitor

ABSTRACT

The present invention relates to a method for treating degenerative neurological disorders in a subject in need thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising an acid sphingomyelinase (ASM) activity inhibitor or expression inhibitor as an active ingredient.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 15/994,934 filed May 31, 2018, which is acontinuation-in-part of U.S. patent application Ser. No. 14/889,293filed Nov. 5, 2015, which is a national phase application under 35U.S.C. § 371, of PCT/KR2014/004025, filed May 7, 2014, which claims thebenefit of priority to Korean Patent Application No. 10-2013-0051279,filed on May 7, 2013, the contents of which are incorporated byreference herein in their entirety.

BACKGROUND Field

Exemplary embodiments relate to a method for treating degenerativeneurological disorders in a subject in need thereof, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a composition comprising an acid sphingomyelinase (ASM)activity inhibitor or expression inhibitor as an active ingredient.

Discussion of the Background

Dementia refers to a progressive decline in memory and cognitivefunctioning that interferes with daily life, and may be largely dividedinto vascular dementia and Alzheimer's disease. Vascular dementia mainlycorresponds to the case where stroke or cerebral infarction, etc. occursby thrombus formed in blood vessels, and is known to be the onset ofsymptoms such as memory loss, etc. caused by damage to neighboring braincells. On the other hand, Alzheimer's disease (AD), which accounts for asignificantly greater part of dementia than vascular dementia, is aprogressive brain disorder that slowly weakens memory, changespersonality, and destroys thinking skills. Most patients of Alzheimer'sdisease die of pneumonia, etc. within 8 to 10 years. Worldwide, 3.5 to10% of the elderly people over the age of 65 suffer from this disease,and there are an estimated 4 million patients only in the US. Socialcosts incurred to treat this disease reach US$100 billion every yearonly in the US, making Alzheimer's disease a signature disease of oldage.

The pathogenesis of Alzheimer's disease known until now is liberatingβ-amyloid from amyloid precursor protein (APP), generating insolubleamyloid plaque by having the liberated β-amyloid cohered, causingdegeneration of neural cells by cohesion of β-amyloid and generation ofamyloid plaques, and inducing generation of secondary neurofibrillarytangle as a result. As such, it has been found out that the accumulationof β-amyloid in brain tissue and neural toxicity accompanied therefromactivate as very important causes of Alzheimer's disease, andaccordingly, research is focused on substances, like BACE-1 inhibitor,that have an effect of inhibiting the generation of β-amyloid,inhibiting cohesion, or inhibiting toxicity, which having less sideeffects, over the world. β-amyloid is a fragment of amyloid precursorprotein generated when APP, an amyloid precursor protein, receivesproteolytic enzymes such as gamma-secretase and beta-secretase. Thebeta-secretase enzyme which plays the most important role in generatingβ-amyloid is generally referred to as BACE, and two types of BACE, i.e.,BACE-1 and BACE-2, etc. are known. Among them, BACE-1 has most activity(about 90%) of beta-secretase, and thus is known to play a much moreimportant role than BACE-2 in generating β-amyloid.

Also, according to recent epidemiologic studies, it has been reportedthat risk factors for cerebrovascular diseases such as high bloodpressure, diabetes, hyperlipidemia and cardiac disorders have increasedthe occurrence of Alzheimer's disease as well as vascular dementia. Fromthe modern medical point of view on cognitive impairment caused byAlzheimer's disease (AD), extensive degeneration and loss of cholinergicneurons in the brain are considered as the leading cause of cognitivedecline, and as a means to overcome this problem, most studies aim todevelop drugs that can partly recover impaired cognitive functioning byincreasing the activity of the cholinergic nervous system leftundamaged.

Recently, four drugs (tacrine, rivastigmine, donepezil, and galantamine)have been approved by the U.S. Food and Drug Administration (FDA) forthe treatment of Alzheimer's disease, and they are all so-calledacetylcholinesterase inhibitors which intend to dramatically improvecognitive functioning by inhibiting the activity of acetylcholinesteraseenzymes. Until now, acetylcholinesterase inhibitors are the only drugsapproved as a therapeutic agent of Alzheimer disease. However, thesedrugs have disadvantages such that they only present a temporary reliefof symptoms in some Alzheimer's patients (40-50%), and the efficacy doesnot last long. Also, although the drug has to be taken for a long periodof time due to the characteristic of the disease, theacetylcholinesterase inhibitors developed until now had problems suchthat they accompanied a number of side effects including liver toxicity.That is, the therapeutic agents developed until now only temporarilyrelieved the symptoms, and thus development of drugs fundamentallytreating the disease or inhibiting the progress of the disease isurgently required.

Meanwhile, sphingolipid metabolism controls signal transduction ofnormal cells, and ASM, an enzyme controlling sphingolipid metabolism, isa protein expressed in almost all cell types, and has an important rolein sphingolipid metabolism and membrane turnover. The ASM is mainlylocated within the endosomal/lysosomal compartment, and when there is acellular stress response, it is transported outside the cell membrane.ASM increases in various diseases such as Wilson's disease, diabetes,cystic fibrosis, emphysema, etc., and may have a significant correlationwith the onset of the diseases. However, despite the above role of ASM,currently there is little progress in studies on the relationshipbetween ASM and Alzheimer's disease.

In this regard, the present inventors found ASM as a pathogenesis ofAlzheimer's disease and completed the present invention by confirmingthat when ASM is partially removed in an Alzheimer's disease modelmouse, that is when ASM is inhibited therein, such as when anAlzheimer's disease model mouse with a partial removal of ASM is in aparabiotic union with an Alzheimer's disease model mouse, or when anAlzheimer's disease model mouse is injected with the serum of an mousefrom which ASM gene has been removed, the deposition of β-amyloid in thebrain tissue is inhibited and the ability to learn and remember isimproved.

SUMMARY

An exemplary embodiment discloses a method for treating a degenerativeneurological disorder in a subject in need thereof, comprisingadministering to a subject a therapeutically effective amount of acomposition comprising an acid sphingomyelinase (ASM) activity inhibitoror expression inhibitor as an active ingredient.

An exemplary embodiment further discloses a method for treatingdepression in a subject in need thereof, comprising administering to asubject a therapeutically effective amount of a composition comprisingan acid sphingomyelinase (ASM) expression inhibitor as an activeingredient, wherein the ASM expression inhibitor comprises a miRNA whichcomplementarily binds to mRNA of ASM gene and comprises at least anucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 3, and SEQ ID NO: 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view illustrating a process for manufacturingAPP/PS1/ASM^(+/−) mice;

FIG. 2 is a view illustrating the ASM concentration levels in theplasma, brain tissue and fibroblast of Alzheimer's disease model mice(APP/PS1) and mice with partial removal of ASM in the Alzheimer'sdisease model mice (APP/PS1/ASM^(+/−)) (left: plasma; middle: braintissue; right: fibroblast);

FIG. 3 is a view confirming the deposition of β-amyloid in the cerebralcortex in the brain tissues of APP/PS1 mice and APP/PS1/ASM^(+/−) miceusing thioflavin S dye;

FIG. 4 is a view confirming the deposition of β-amyloid in thehippocampus in the brain tissues of APP/PS1 mice and APP/PS1/ASM^(+/−)mice using thioflavin S dye;

FIG. 5 is a view confirming the deposition of β-amyloid in the braintissues of APP/PS1 mice and APP/PS1/ASM^(+/−) mice usingimmunofluorescence;

FIG. 6 is a view confirming the deposition of β-amyloid in the braintissues of APP/PS1 mice and APP/PS1/ASM^(+/−) mice using ELISA;

FIGS. 7 and 8 are views confirming the effect of improving the abilityto learn and remember of APP/PS1/ASM^(+/−) mice using Morris water maze(MWM) test;

FIGS. 9 and 10 are views confirming the effect of improving the abilityto remember of APP/PS1/ASM^(+/−) mice using fear conditioning test;

FIG. 11 is a view confirming the expression level of autophagy-relatedprotein in the tail fibroblast from WT mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice using Western blotting;

FIG. 12 is a view confirming the expression level of autophagy-relatedprotein in the tail fibroblast from WT mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice using densitometric quantification;

FIG. 13 is a view confirming the expression level of autophagy-relatedprotein in the brain tissues of WT mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice using Western blotting;

FIG. 14 is a view confirming the expression level of autophagy-relatedprotein in the brain tissues of WT mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice using densitometric quantification;

FIG. 15 is a view confirming the proteolytic activity in the tailfibroblast from WT mice, APP/PS1 mice and APP/PS1/ASM^(+/−) mice;

FIG. 16 is a view observing the brain tissues of WT mice, APP/PS1 miceand APP/PS1/ASM^(+/−) mice using transmission electron microscope (TEM);

FIG. 17 is a view confirming the expression level of autophagy-relatedprotein when treating human fibroblast with ASM (1 μM to 10 μM) usingWestern blotting;

FIG. 18 is a view confirming the expression level of autophagy-relatedprotein when treating human fibroblast with ASM (1 μM to 10 μM) usingdensitometric quantification;

FIG. 19 is a view confirming the expression level of autophagy-relatedprotein when treating human fibroblast with M6P using Western blottingand densitometric quantification;

FIG. 20 is a view illustrating the conversion rate from LC3-I to LC3-IIwhen treating human fibroblast with ASM in the presence or absence ofNH₄Cl using Western blotting;

FIG. 21 is a view illustrating the conversion rate from LC3-I to LC3-IIwhen culturing human fibroblast in a serum-free medium or completemedium and treating it with NH₄Cl;

FIG. 22 is a view illustrating the conversion rate from LC3-I to LC3-IIwhen culturing human fibroblast in a serum-free medium or completemedium and treating it with ASM;

FIG. 23 is a view illustrating a test design for verifying the effect oftreating Alzheimer's disease by administering AMI into APP/PS1 mice;

FIG. 24 is a view illustrating the ASM concentration in the serum andbrain tissues of the mice when administering AMI into APP/PS1 mice;

FIG. 25 is a view confirming the deposition of β-amyloid in the braintissues (cerebral cortex and hippocampus) of mice when administering AMIinto APP/PS1 mice;

FIG. 26 is a view confirming the effect of improving the ability toremember when administering AMI into APP/PS1 mice and performing Morriswater maze (MWM) test;

FIG. 27 is a view illustrating a test design for verifying the effect oftreating Alzheimer's disease using parabiotic union system [isochronic(APP/PS1-APP/PS1: parabiotic union between APP/PS1 mice, which areAlzheimer's disease model mice), heterochronic I (APP/PS1-ASM^(+/−):parabiotic union between APP/PS1 mice and ASM^(+/−) mice), heterochronicII (APP/PS1-WT: parabiotic union between APP/PS1 mice and wild typemice)];

FIG. 28 is a view illustrating the ASM concentration in the serum andbrain tissues of parabiotic union mice;

FIG. 29 is a view confirming the deposition of β-amyloid in the braintissues (cerebral cortex and hippocampus) of parabiotic union mice;

FIG. 30 is a view illustrating the expression level for each protein inthe brain tissues of parabiotic union mice using Western blotting;

FIG. 31 is a view illustrating a test design for verifying the effect oftreating Alzheimer's disease using serum injection;

FIG. 32 is a view illustrating the ASM concentration in serum and braintissues of APP/PS1 mice provided with serum of each mouse;

FIG. 33 is a view confirming the deposition of β-amyloid in the braintissues (cerebral cortex and hippocampus) of APP/PS1 mice provided withserum of each mouse;

FIG. 34 is a view illustrating the expression level for each protein inthe brain tissue of APP/PS1 mice provided with serum of each mouse usingWestern blotting;

FIG. 35 is a view confirming the effect of improving the ability toremember of APP/PS1 mice provided with serum of each mouse using Morriswater maze (MWM) test;

FIG. 36 is a view confirming the effect of improving the ability toremember of APP/PS1 mice provided with serum of each mouse using fearconditioning test;

FIG. 37 is a schematic diagram of the role of ASM to a pathogenesis ofAlzheimer's disease;

FIG. 38 illustrates changes in ASM activity after treatment of anti-ASMantibody (ASM-ab: SMPD1 antibody) on fibroblasts (PS1 fibroblast,control) of Alzheimer's patients with increased ASM activity(n=3/group);

FIG. 39A illustrates the results of Western blotting analysis of theexpression of autophagy-related proteins after treating theASM-inhibiting antibody (ASM-ab: SMPD1 antibody) on a fibroblast ofAlzheimer's disease. (PS1 fibroblast);

FIG. 39B illustrates the quantification results of the Western blottinganalysis of FIG. 39A (n=3/group);

FIG. 40 is a diagram illustrating the outline of an experiment performedto examine the effect of ASM inhibition on Alzheimer's disease throughASM-microRNA (Smpd1 miR RNAi) injection;

FIG. 41A illustrates the changes in ASM concentration in mouse serumafter injection of Control miR RNAi or Smpd1 miR RNAi into anAlzheimer's animal model (n=3-4/group) (WT: wild type, AD: Alzheimer'sanimal model mouse (APP/PS1 mouse));

FIG. 41B illustrates the changes in ASM concentration in mouse braintissues after injection of Control miR RNAi or Smpd1 miR RNAi into anAlzheimer's animal model (n=3-4/group) (WT: wild type, AD: Alzheimer'sanimal model (APP/PS1 mouse));

FIG. 42A illustrates the immunofluorescence staining images usingthioflavin S (ThioS, fibrillary amyloid beta plaque detection) in thebrain cortex of Alzheimer's animal model injected with Control miR RNAior Smpd1 miR RNAi and the quantification results of the area occupied bya fibrillary amyloid beta plaque detected by Thioflavin S. (N=3/group)(WT: wild type, AD: Alzheimer's animal model (APP/PS1 mouse));

FIG. 42B illustrates the immunofluorescence staining images usingthioflavin S (ThioS, fibrillary amyloid beta plaque detection) in thebrain hippocampus of Alzheimer's animal model injected with Control miRRNAi or Smpd1 miR RNAi and the quantification results of the areaoccupied by a fibrillary amyloid beta plaque detected by Thioflavin S.(N=3/group) (WT: wild type, AD: Alzheimer's animal model (APP/PS1mouse));

FIG. 43A illustrates the immunofluorescent staining image showing thedegree of accumulation of Aβ40 in the brain cortex and hippocampus of anAlzheimer's animal model injected with Control miR RNAi or Smpd1 miRRNAi and the result of quantifying the degree of accumulation(n=3/group) (WT: wild type, AD: Alzheimer's animal model (APP/PS1mouse));

FIG. 43B illustrates the immunofluorescent staining image showing thedegree of accumulation of Aβ42 in the brain cortex and hippocampus of anAlzheimer's animal model injected with Control miR RNAi or Smpd1 miRRNAi and the result of quantifying the degree of accumulation.(n=3/group) (WT: wild type, AD: Alzheimer's animal model (APP/PS1mouse));

FIG. 44A illustrates the change of the escape time during the MWM testperiod. The MWM test was performed to assess the ability of learning andmemory on wild-type mice (n=6), Alzheimer's animal model injected withControl miR RNAi (n=6), Alzheimer's animal model injected with Smpd1 miRRNAi (n=6) (WT: wild type, AD: Alzheimer's animal model (APP/PS1mouse));

FIG. 44B illustrates the time period spent during which the mice stayedon the target platform on the 11th day of the MWM test;

FIG. 44C illustrates the number of times the mouse entered the targetarea of the target platform on the 11th day of the MWM test;

FIG. 45A illustrates the results of quantifying the percentage ofastrocytes (marker GFAP) in the brain cortex of the Alzheimer's animalmodel injected with Control miR RNAi or Smpd1 miR RNAi, and thewild-type mice (n=3/group, WT: wild type, AD: Alzheimer's animal model.(APP/PS1 mouse));

FIG. 45B illustrates the results of quantifying the percentage ofmicroglial cells (marker Iba-1) in the brain cortex of the Alzheimer'sanimal model injected with Control miR RNAi or Smpd1 miR RNAi, and thewild-type mice. (n=3/group, WT: wild type, AD: Alzheimer's animal model.(APP/PS1 mouse));

FIG. 45C illustrates the results of evaluating mRNA expression levels ofpro-inflammatory markers (TNF-alpha, IL-1beta, IL-6) in the brain cortexof the Alzheimer's animal model injected with Control miR RNAi or Smpd1miR RNAi, and the wild-type mice (n=3/group, WT: wild type, AD:Alzheimer's animal model. (APP/PS1 mouse));

FIG. 46A illustrates the results of Western blotting analysis of theexpression of autophagy-related proteins in the brain cortex of theAlzheimer's animal model injected with Control miR RNAi or Smpd1 miRRNAi, and the wild-type mice (WT: wild type, AD: Alzheimer's animalmodel (APP/PS1 mouse)); and

FIG. 46B illustrates the quantification results of the Western blottinganalysis of FIG. 46A (n=3/group).

FIG. 47A is a diagram illustrating a process of injecting Control miRRNAi or Smpd1 miR RNAi into a depression-induced animal model.

FIG. 47B shows changes in ASM concentration in plasma of the mice ofFIG. 47A (n=4/group) (WT: wild type, WT/RSD: depression induced mousemodel).

FIG. 48A shows the result of the Open field test showing the degree ofrecovery of depression behavior pattern in the depression animal modelinjected with Control miR RNAi or Smpd1 miR RNAi (n=3/group) (WT: wildtype, WT/RSD: depression induced mouse model).

FIG. 48B shows the result of the Dark & Light test showing the degree ofrecovery of depression behavior pattern in the depression animal modelinjected with Control miR RNAi or Smpd1 miR RNAi (n=3/group) (WT: wildtype, WT/RSD: depression induced mouse model).

FIG. 48C shows the result of the Tail suspension test showing the degreeof recovery of depression behavior pattern in the depression animalmodel injected with Control miR RNAi or Smpd1 miR RNAi (n=3/group) (WT:wild type, WT/RSD: depression induced mouse model).

FIG. 48D shows the result of the Force swim test showing the degree ofrecovery of depression behavior pattern in the depression animal modelinjected with Control miR RNAi or Smpd1 miR RNAi (n=3/group) (WT: wildtype, WT/RSD: depression induced mouse model).

DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treatingdegenerative neurological disorders, including an ASM (acidsphingomyelinase) activity inhibitor or expression inhibitor as anactive ingredient.

The composition includes a pharmaceutical composition or a foodcomposition.

According to the present invention, when ASM was inhibited inAlzheimer's disease model mice, for example, in the case of aAlzheimer's disease model mice in which ASM gene is partially deficient,in the case of a case in which a Alzheimer's disease model mice and aASM gene-partially deficient mice were parabiotically unioned, and inthe case of a Alzheimer's disease model mice injected with the sera froma ASM gene-deficient mouse, the deposition of β-amyloid in the braintissue is inhibited and the ability to learn and remember is improved,and the present invention confirms such superb effects. Accordingly, ASMinhibitor may be effectively used to prevent or treat degenerativeneurological disorders including Alzheimer's disease.

In the present invention, ASM (acid sphingomyelinase, gene Smpd1) is notlimited to a specific biological origin. The amino acid sequence of ASMis not particularly limited to a specific amino acid sequence as long asit has an amino acid sequence of a polypeptide known as ASM, while thenucleic acid sequence of a gene encoding ASM (including all transcriptsfrom ASM gene, such as mRNA and the like) is also not particularlylimited. As a preferable example, human ASM may comprise an amino acidsequence known as Genbank Accession No. NP_000534.3, NP_001007594.2,NP_001305016.1, NP_001305017.1, and the like, while its gene (or mRNA)sequence is known as Genbank Accession No. NP_000543.4, NP_001007593.2,NP_001318087.1, NP_001318088.1, and the like.

The ASM activity inhibitor according to the present invention may be atleast one selected from a group consisting of a compound, a peptide, apeptide mimetic, a substrate analogue, an aptamer, and an antibody,specifically binding to ASM protein, but is not limited thereto.

The peptide mimetics inhibit the binding domain of ASM protein, thusinhibiting the activity of ASM protein. The peptide mimetics may bepeptides or non-peptides and may include amino acids linked bynon-peptide bonds such as psi bonds (Benkirane, N., et al. J. Biol.Chem., 271:33218-33224, 1996). Moreover, the peptide mimetics may be“conformationally constrained” peptides, cyclic mimetics, or cyclicmimetics including at least one exocyclic domain, a link moiety (linkingamino acid) and an active region. The peptide mimetics are constructedto resemble secondary structural features of Ubiquitin-AssociatedProtein 2 (UBAP2) and may mimic inhibitory features of macro moleculessuch as antibody (Park, B. W. et al. Nat Biotechnol 18, 194-198, 2000)or water soluble receptors (Takasaki, W. et al. Nat Biotechnol 15,1266-1270, 1997). These peptides represent novel small molecule that mayact with potency equivalent to the natural antagonist (Wrighton, N. C.et al. Nat Biotechnol 15, 1261-1265, 1997).

The aptamer is a single-stranded DNA or RNA molecule and may be obtainedby isolating oligomers that bind to specific chemical molecules orbiological molecules with high affinity and specificity by anevolutionary method using an oligonucleotide library called systematicevolution of ligands by exponential enrichment (SELEX) (C. Tuerand L.Gold, Science 249, 505-510, 2005; A. D. Ellington and J. W. Szostak,Nature 346, 818-822, 1990; M. Famulok, et. al., Acc. Chem. Res. 33,591-599, 2000; D. S. Wilson and Szostak, Annu. Rev. Biochem. 68,611-647, 1999). The aptamer may specifically bind to a target toregulate its activity and may inhibit the function of the target bybinding, for example.

The antibody specifically and directly binds to the ASM to effectivelyinhibit its activity. Preferably, a polyclonal antibody or monoclonalantibody may be used as the antibody that specifically binds to the ASM.The antibody that specifically binds to the ASM may be prepared by amethod known to those skilled in the art, and a commercially availableASM antibody may be purchased and used. The antibody may be prepared byinjecting the ASM protein as an immunogen into an external hostaccording to a conventional method known to those skilled in the art.The external host may include mammals such as mice, rats, sheep,rabbits, etc. The immunogen may be injected intramuscularly,intraperitoneally, or subcutaneously, and generally may be injected withan adjuvant to enhance antigenicity. Blood samples may be taken from theexternal host at regular intervals and serum exhibiting titer andspecificity to the antigen may be collected to separate an antibodytherefrom.

The ASM expression inhibitor according to the present invention may beat least one selected from a group consisting of an antisensenucleotide, small hairpin RNA (shRNA), small interfering (siRNA),microRNA (miRNA) and ribozyme, complementarily binding to mRNA of an ASMgene or a gene promoting expression of ASM, but is not limited thereto.The antisense nucleotide, shRNA, siRNA or miRNA of the present inventionis not particularly limited in its nucleotide sequence and its length aslong as it has an activity of suppressing the expression of the ASM gene(i.e., Smpd1). As an example, the antisense nucleotide, shRNA, siRNA ormiRNA of the present invention may comprise a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 6, butis not limited thereto.

The siRNA is composed of a sense sequence of 15 to 30-mers selected fromthe mRNA sequence of a gene that encodes the ASM protein and anantisense sequence complementarily binding to the sense sequence. Here,preferably, the sense sequence may be composed of about 25 nucleotides,but is not particularly limited thereto.

As used herein, the miRNA (microRNA) refers to a short non-coding RNAderived from an endogenous gene, which acts as a post-transcriptionalregulator of gene expression. miRNA acts as a post-transcriptionalregulator of gene expression by forming a base pair with the mRNA of atarget gene. The miRNA according to the present invention is notparticularly limited in its nucleotide sequence and its length as longas it has an activity of suppressing the expression of the ASM gene(i.e., Smpd1). As an example, the miRNA according to the presentinvention may comprise a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 6. As another example, themiRNA of the present invention may consist of or essentially consist ofany one of the nucleotide sequences selected from the group consistingof SEQ ID NOS: 1 to 6.

As defined by Watson-Crick base pairs, the antisense nucleotide ishybridized with a complementary sequence of DNA, immature-mRNA ormature-mRNA to interrupt the transmission of genetic information as aprotein in DNA. A target sequence specific antisense nucleotide isexceptionally multi-functional. The antisense nucleotide is a long chainof monomers, which favors hybridization to a target RNA sequence.Numbers of reports have recently been made to prove the utility of anantisense nucleotide as a biochemical tool in study of a target protein(Rothenberg et al., J. Natl. Cancer Inst., 81:1539-1544, 1999). Greatprogress has been made in the fields of oligonucleotide chemistry andnucleotide synthesis having improved cell adhesion of oligonucleotide,target binding affinity and resistance against nuclease, suggesting thatan antisense nucleotide might be considered a new form of an inhibitor.

The degenerative neurological disorder according to the presentinvention includes Alzheimer's disease, Parkinson's disease, progressivesupranuclear palsy, multiple system atrophy, olivopontocerebellaratrophy (OPCA), Shy-Drager syndrome, Striatonigral degeneration,Huntington's disease, Amyotrophic lateral sclerosis (ALS), essentialtremor, cortico-basal ganglionic degeneration, diffuse Lewy bodydisease, Parkinson-ALS-dementia complex of Guam, Pick's disease,ischemia, and cerebral infarction, and depression, but is not limitedthereto.

The composition of the present invention may include, together with theASM activity inhibitor or expression inhibitor, at least one of a knownactive ingredient having an effect of inhibiting ASM expression oractivity, or a known active ingredient having an effect of treatingdegenerative neurological disorders.

Also, the pharmaceutical composition of the present invention may beadministered orally or parenterally (e.g., applied intravenously,subcutaneously, intraperitoneally or topically) according to theintended use, and the dosage may vary according to a patient's weight,age, gender, health condition, diet, administration time, administrationmethod, administration period or interval, excretion rate,constitutional specificity, nature of formulation, etc. The dosage ofthe ASM expression inhibitor or activity inhibitor of the presentinvention is about 0.001 to 1000 mg/kg per day, preferably about 0.1 to500 mg/kg per day, but this may vary depending on the clinical testresult. Preferably, the pharmaceutical composition of the presentinvention may be administered once or several times a day.

The pharmaceutical composition of the present invention may beformulated in a variety of formulations for administration. Theexcipients that may be included in the present invention are non-toxicinert pharmaceutically suitable solid, semi-solid or liquid formulationauxiliaries of any type, for example, fillers, weighting agents,binders, wetting agents, disintegrating agents, dispersing agents,surfactants or diluents, etc.

The pharmaceutical composition of the present invention may beformulated in the form of tablets, coated tablets, capsules, pills,granules, suppositories, solutions, suspensions and emulsions, pastes,ointments, gels, creams, lotions, powders or sprays.

The composition of the present invention may be added to dietarysupplements for the improvement of degenerative neurological disorders.When using the ASM expression inhibitor or activity inhibitor of thepresent invention as a food additive, the active ingredient may be addedas it is or together with other food or food ingredients, and it may besuitably used according to a conventional manner. The amount of activeingredient added may be determined properly according to the purpose ofuse (preventive, health or therapeutic purposes). In general, whenmanufacturing food or beverage, the active ingredient of the presentinvention is added in an amount of 15% by weight or less to the rawmaterial, preferably in an amount of 10% by weight or less. However, forhealth and hygiene purposes, or for long-term intake for the purpose ofhealth control, the amount of active ingredient may be equal to or lessthan the above range, and since there is no problem in terms of safety,the active ingredient may be used in an amount greater than or equal tothe above range.

There is no particular limitation in the type of food. Examples of thefood to which this substance may be added include meat, sausages, bread,chocolate, candies, snacks, confectionery, pizza, ramen, other noodles,gum, dairy products including ice cream, soup, beverages, tea, drinks,alcohol drinks, and vitamin complexes, etc. That is, food may compriseall kinds of dietary supplements in the conventional sense.

The health beverage composition of the present invention may includeadditional ingredients such as various flavoring agents or naturalcarbohydrates, etc., like other beverages. The natural carbohydratesabove may be monosaccharides such as glucose and fructose, disaccharidessuch as maltose and sucrose, polysaccharides such as dextrin andcyclodextrin, and sugar alcohols such as xylitol, sorbitol anderythritol, etc. Natural sweeteners such as thaumatin and steviaextract, and synthetic sweeteners such as saccharin and aspartame, etc.may be used as sweeteners. The ratio of natural carbohydrate isgenerally in the range of about 0.01 to 0.20 g per 100 g of thecomposition of the present invention, and preferably in the range ofabout 0.04 to 0.10 g.

In addition to the above, the composition of the present invention mayinclude various nutrients, vitamins, electrolytes, flavoring agents,coloring agents, pectic acid and its salts, alginic acid and its salts,organic acid, protective colloidal thickeners, pH adjusting agents,stabilizers, preservatives, glycerin, alcohol, carbonating agents usedin carbonated beverages, etc. Further, the composition of the presentinvention may include pulp for the production of natural fruit juice,fruit juice beverage and vegetable beverage. These ingredients may beused independently or in combination with other ingredients. The ratioof the additive is not so important but is generally selected from arange of 0.01 to 0.20 parts by weight with respect to 100 parts byweight of the composition of the present invention.

The term “comprising” of the present invention is used synonymously with“including” or “characterized” and does not exclude additional componentelements or method steps, etc., not mentioned in the composition ormethod. The term “consisting of” is intended to exclude additionalelements, steps or components, etc., not otherwise mentioned. The term“essentially consisting of” is intended to encompass component elementsor steps, etc., which, in addition to the described component elementsor steps, do not materially affect the active ingredient (for instance,ASM inhibitor in the present invention) underlying properties in thescope of the composition or method.

Also, the present invention provides a method for preventing or treatingdegenerative neurological disorders, including administering to asubject in need thereof a therapeutically effective amount of thecomposition.

Specifically, the present invention provides a method for treating adegenerative neurological disorders in a subject in need thereof,comprising administering to a subject a therapeutically effective amountof a composition comprising an ASM (acid sphingomyelinase) activityinhibitor or expression inhibitor as an active ingredient.

Also, the present invention provides a method for treating adegenerative neurological disorders in a subject in need thereof,comprising administering to a subject a therapeutically effective amountof a composition consisting of an ASM (acid sphingomyelinase) activityinhibitor or expression inhibitor.

Also, the present invention provides a method for treating adegenerative neurological disorders in a subject in need thereof,comprising administering to a subject a therapeutically effective amountof a composition essentially consisting of an ASM (acidsphingomyelinase) activity inhibitor or expression inhibitor.

Further, an embodiment of the present invention provides a method fortreating depression in a subject in need thereof, comprisingadministering to a subject a therapeutically effective amount of acomposition comprising an acid sphingomyelinase (ASM) expressioninhibitor as an active ingredient, wherein the ASM expression inhibitorcomprises a miRNA which complementarily binds to mRNA of ASM gene andcomprises at least a nucleic acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5.

The term “treatment or treating” as used herein is a concept involvinginhibition, elimination, alleviation, relief, amelioration and/orprevention of a disease itself, or symptoms or conditions caused by thedisease.

The “effective amount” as used herein refers to an amount that, whenadministered to an individual, represents an improvement, treatment, orprevention effect of degenerative neurological disorders. It is obviousto those skilled in the art that the therapeutically effective amountmay be determined within the scope of sound medical judgment.Preferably, the specific therapeutically effective amount for aparticular patient may vary depending on a variety of factors includingthe type and degree of a desired reaction, the specific compositionincluding the use of any other agents according to the intended use, thepatient's age, weight, general health condition, gender, diet,administration time, administrate route and excretion rate of thecomposition, duration of treatment, other drugs used in combination orcoincidentally with the specific composition, and like factors wellknown in the medical arts. Therefore, preferably, the effective amountof the composition suitable for the purpose of the present invention isdetermined in consideration of the foregoing.

In addition, optionally, the composition of the present invention may beadministered in combination with a known therapeutic agent fordegenerative neurological disorders to increase the effect of treatingdegenerative neurological disorders.

The term “subject” refers to an animal, preferably a mammal whichespecially includes a human, while including animal-derived cells,tissues, organs and the like. The subject may be a patient in need ofthe above mentioned effect.

The present invention is applicable to any mammal, with a degenerativeneurological disorder as described in the above-mentioned “subject”.Here, the mammals include human, primates, and livestock animals such ascows, pigs, sheep, horses, dogs, cats, etc.

Also, the present invention provides a method for screening a substancefor preventing or treating degenerative neurological disorders,including 1) treating a biological sample with a candidate substance,and 2) measuring the change in expression amount of mRNA or protein ofASM (acid sphingomyelinase) from the biological sample in step 1).

The biological sample in the step 1) may include blood, urine, saliva ortissue, etc. of animals with degenerative neurological disorders, but isnot limited thereto.

The method for measuring the change in expression amount of mRNA in thestep 2) includes reverse transcription polymerase chain reaction(RT-PCR), competitive RT-PCR, realtime RT-PCR, RNase protection assay(RPA), Northern blotting, and DNA chip, etc., but is not limitedthereto.

The method for measuring the change in expression amount of protein inthe step 2) includes Western blotting, enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlonyimmunodiffusion, rocket immunoelectrophoresis, immunohi stostaining,immunoprecipitation assay, complement fixation assay, fluorescenceactivated cell sorter (FACS) and protein chip, etc., but is not limitedthereto.

Hereinafter, preferred Examples will be provided to facilitateunderstanding of the present invention. However, the following Examplesare provided for better understanding of the present invention only, andthe scope of the present invention is not limited by the followingExamples.

Example 1. Confirmation on the Effect of ASM Inhibition on the Treatmentof Alzheimer's Disease in ASM (Acid Sphingomyelinase) Mutant Mice 1-1.Preparation of ASM Mutant Mice

An experiment was conducted using APP/PS1 (APP/presenilin) double mutantmice and APP/PS1/ASM^(+/−) triple mutant mice (with partial geneticremoval of ASM), which are test animal models of Alzheimer's disease.

The animal test conducted was approved by the Kyungpook NationalUniversity Institutional Animal Care and Use Committee (IACUC).Transgenic mouse lines overexpressing APPswe (hAPP695swe) or PS1(presenilin-1M146V) mutations were used onto C57BL/6 mice (CharlesRiver, UK) [hereinafter, APP mice: mice overexpressing APPswe, PS1 mice:overexpressing presenilin-1M146V; GlaxoSmithKline]. ASM^(+/−) mice (onlyone of a pair of ASM genes is removed) were crossed with APP/PS1 mice toprepare APP/PS1/ASM^(+/−) mice.

Detailed process is illustrated in FIG. 1.

1-2. Confirmation on ASM Concentration Level in ASM Mutant Mice

The ASM concentration levels were measured in the plasma, brain tissueand fibroblast of nine-month old wild type (WT) mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice prepared in Example 1-1. More specifically, 3 μlof plasma, brain tissue and fibroblast samples from each mouse weremixed with an ASM activity buffer, and stored at 37° C. The hydrolysisreaction was completed by adding 114 μl of ethanol to the mixedsolution, and then the mixed solution was centrifuged. 30 μl of thesupernatant was transferred into a glass vial, and then 5 μl was appliedto the UPLC system. The ASM concentration level was quantified incomparison with a bodipy (aminoacetaldehyde) combined with sphingomyelinand ceramide. The sphingomyelin and ceramide levels were extracted andquantified according to a known method, by extracting lipid from thesample, resuspending the dried lipid extract in 25 μl of 0.2% IgepalCA-630 (Sigma-Aldrich), and quantifying the concentration level of eachlipid using the UPLC system. The results are illustrated in FIG. 2(left: plasma; middle: brain tissue; right: fibroblast).

As illustrated in FIG. 2, it is confirmed that the ASM concentrationlevels in the plasma, brain tissue and fibroblast of APP/PS1/ASM^(+/−)mice decreased remarkably, as compared to the ASM concentration levelsin the plasma, brain tissue and fibroblast of APP/PS1 mice.

1-3. Confirmation on Inhibition of Deposition of β-Amyloid in BrainTissue of ASM Mutant Mice

As confirmed in Example 1-2, in order to confirm how the decrease in ASMconcentration level in APP/PS1/ASM^(+/−) mice affects Alzheimer'sdisease in a pathological aspect, the deposition level of β-amyloid inthe brain tissue was analyzed.

First, the cerebral cortex and hippocampus tissue of each mouse preparedin Example 1-1 were isolated, and then a tissue fragment was obtainedand this was dyed with thioflavin S according to a conventional knownmethod. The results are illustrated in FIG. 3 (cerebral cortex) and FIG.4 (hippocampus).

As illustrated in FIG. 3 and FIG. 4, it is confirmed that Aβ40 and Aβ42deposited in the brain tissue of APP/PS1/ASM^(+/−) mice decreasedremarkably, as compared to the brain tissue of APP/PS1 mice.

Also, immunofluorescence was performed according to a conventional knownmethod using anti-20G10 (mouse, 1:1000) antibody against Aβ42, anti-G30(rabbit, 1:000) antibody against Aβ40, anti-Iba-1 (rabbit, 1:500, Wako)antibody, anti-GFAP (rabbit, 1:500, DAKO) antibody and anti-activecaspase3 (rabbit, 1:50, Chemicon) antibody. Tissue fragments wereobserved using a confocal laser scanning microscope equipped withFluoview SV1000 imaging software (Olympus FV1000, Japan) or an OlympusBX51 microscope, and the percentage of the area of the dyed area withrespect to the area of the entire tissue was quantified using Metamorphsoftware (Molecular Devices).

Also, β-amyloid deposition was confirmed using commercially availableELISA kits (Biosource). More specifically, hemispheres of the brain ofeach mouse were homogenized in a buffer containing 0.02 M of guanidine.Thereafter, ELISA was performed for Aβ40 and Aβ42 according to themanufacturer's instructions.

The results are illustrated in FIG. 5 (immunofluorescence) and FIG. 6(ELISA).

As illustrated in FIG. 5 and FIG. 6, it is confirmed that Aβ40 and Aβ42deposited in the brain tissue of APP/PS1/ASM^(+/−) mice decreasedremarkably, as compared to the brain tissue of APP/PS1 mice.

1-4. Confirmation on Improvement of Ability to Learn and Remember in ASMMutant Mice

In order to confirm the effect of improving the ability to learn andremember in APP/PS1/ASM^(+/−) mice prepared in Example 1-1, a Morriswater maze (MWM) test was performed according to a conventional knownmethod.

More specifically, wild type mice, APP/PS1/ASM^(+/−) mice, ASM^(+/−)mice and APP/PS1 mice were used. The mice were given four trials per dayfor 10 days to learn the task, and on day 11, the mice were given aprobe trial in which the platform was removed. The escape latency duringthe test period and the time spent in the target platform on day 11 weremeasured. The results are illustrated in FIG. 7 and FIG. 8.

As illustrated in FIG. 7 and FIG. 8, APP/PS1 mice did not show anychange in escape latency during the test period, and did not show anydifference in the time spent in the target platform and non-targetplatform, and thus it is confirmed that APP/PS1 mice showed disorder informing spatial memory. In comparison, it is confirmed thatAPP/PS1/ASM^(+/−) mice presented improved ability to learn and rememberto a level similar to wild type mice.

In order to verify the MWM test results, a fear conditioning test wasperformed, which evaluates the ability to learn and remember bycombining environmental context or conditioned stimulus with electricshock according to a conventional known method.

More specifically, wild type mice, APP/PS1/ASM^(+/−) mice, ASM^(+/−)mice and APP/PS1 mice were used. On day 1 of the test, each mouse wasindividually placed into a conditioned chamber, and then after a60-second exploratory period, a tone (10 kHz, 70 dB) was delivered for10 seconds. This served as the conditioned stimulus (CS). The CS wascoterminated with the unconditioned stimulus (US), an electricalfootshock (0.3 mA, 1 s). The CS-US pairing was delivered twice at a20-second intertrial interval. On day 2, each mouse was placed in thefear-conditioning chamber containing the same exact context, but withoutadministration of a CS or footshock. Freezing response was observed for5 minutes. On day 3, each mouse was placed in a test chamber that wasdifferent from the conditioning chamber. After a 60-second exploratoryperiod, the tone was presented for 60 seconds without the footshock.Freezing response was measured to measure fear memory. The results areillustrated in FIG. 9 and FIG. 10.

As illustrated in FIG. 9 and FIG. 10, it is confirmed thatAPP/PS1/ASM^(+/−) mice has improved ability to remember than APP/PS1mice.

Example 2. Confirmation on Effect of ASM Inhibition on Autophagy in ASM(Acid Sphingomyelinase) Mutant Mice 2-1. Confirmation onAutophagy-Related Gene Expression by ASM Inhibition

In order to confirm how genetic ASM inhibition works onautophagy-related pathways of Alzheimer's disease, the tail fibroblastand brain tissue samples from nine-month old wild type mice, APP/PS1mice and APP/PS1/ASM^(+/−) mice prepared in Example 1-1 were analyzed.

More specifically, Western blotting was performed according to aconventional known method using LC3 (rabbit, 1:1000, Cell SignalingTechnologies, 4108S), Beclin-1 (rabbit, 1:1000, Cell SignalingTechnologies, 3738S), p62 (rabbit, 1:1000, Cell Signaling Technologies,5114S), cathepsin D (goat, 1:500, R&D Systems, BAF1029) and β-actin(1:1000, Santa Cruz, SC-1615) antibodies, and densitometricquantification was performed using ImageJ software (US NationalInstitutes of Health). The results are illustrated in FIGS. 11 to 14.

As illustrated in FIGS. 11 to 14, it is confirmed that the conversionfrom LC3-I to LC3-II increased in APP/PS1 mice as compared to WT mice,and that the expression of increased LC3-II decreased inAPP/PS1/ASM^(+/−) mice. Beclin-1 expression did not significantly varybetween the groups. Also, the expression of cathepsin D (lysosomalhydrolase) and p62, which are indicators of autophagic turnover,increased in Alzheimer's patients, and thus this is pathologicallyrelated to Alzheimer's disease. It is confirmed that the expression ofcathepsin D and p62 increased in APP/PS1 mice as compared to WT mice,whereas the increased expression of cathepsin D and p62 decreased inAPP/PS1/ASM^(+/−) mice.

2-2. Evaluation on Proteolytic Activity by ASM Inhibition

The proteolytic activity in the tail fibroblast from nine-month old wildtype mice, APP/PS1 mice and APP/PS1/ASM^(+/−) mice prepared in Example1-1 were confirmed.

In order to label long-lived proteins, pulse-chase experiment wasperformed by giving a pulse with [³H]-leucine (2 μCi/ml) for 48 hours.Labeled cells were washed, and cultured in a complete medium of anenvironment inhibiting autophagy or a serum starvation medium inducingautophagy. Aliquots of the medium were collected at different timeperiod and precipitated with 10% TCA, and then they were filtered with afilm having holes of 0.22 m and the radioactivity was measured toanalyze proteolytic activity. The results are illustrated in FIG. 15.

As illustrated in FIG. 15, when autophagy (culturing in serum starvationmedium) is induced, it is confirmed that proteolytic activity increasedin cells derived from APP/PS1/ASM^(+/−) mice, as compared to cellsderived from WT mice.

2-3. Analysis on Mouse Brain Tissue Using Transmission ElectronMicroscope (TEM)

The brain tissues of nine-month old wild type mice, APP/PS1 mice andAPP/PS1/ASM^(+/−) mice prepared in Example 1-1 were fixed in 3%glutaraldehyde containing phosphate buffer, 0.1M, pH 7.4, and postfixedin Sorensen's phosphate buffer containing osmium tetroxide. Afterdehydration with ethyl alcohol, the tissues were embedded in Epon(Electron Microscopy Sciences). They were cut serially and analyzedusing Transmission Electron Microscope (Tecnai). Images were captured ona digital camera and Xplore3D tomography software. The results areillustrated in FIG. 16.

As illustrated in FIG. 16, it is confirmed that the size and number ofautophagic vacuole (AV) increased in the brain tissue of APP/PS1 mice,and the size and number of vacuole in the brain tissue ofAPP/PS1/ASM^(+/−) mice were observed to be slightly greater than thosein WT mice, but smaller than those in APP/PS1 mice.

Example 3. Confirmation on Effect of ASM Inhibition on Autophagy inHuman Cell 3-1. Confirmation on Change in Autophagy-Related GeneExpression by Recombinant ASM Protein in Human Fibroblast

Human fibroblast acquired from the Coriell Institute, and was culturedin DMEM medium containing 15% FBS at 37° C. and 5% of CO₂. The celllines were treated with recombinant ASM (1 μM to 10 μM), and thenWestern blotting and densitometric quantification were performed in thesame manner as Example 2-1. The results are illustrated in FIG. 17 andFIG. 18.

As illustrated in FIG. 17 and FIG. 18, it is confirmed that conversionfrom LC3-I to LC3-II takes place depending on the concentration ofrecombinant ASM treatment, and there is no significant change inexpression of beclin-1.

3-2. Confirmation on Mechanism of ASM Using M6P (Mannose-6-Phosphate)

In order to confirm how ASM affects autophagy, a test was conducted asfollows. Human fibroblast was treated with ASM alone, or treated withASM in the presence of mannose-6-phosphate (M6P; 10 mM) relating to theaction of placing lysosomal enzyme protein in lysosome, and then theaccumulation of autophagosome was confirmed using Western blotting anddensitometric quantification. The results are illustrated in FIG. 19.

As illustrated in FIG. 19, it is confirmed that when M6P is used toinhibit ASM absorption by lysosomes, the conversion to LC3-II decreased,and accordingly the accumulation of ASM-induced autophagosome decreased.

In order to confirm whether ASM increases the formation rate ofautophagosome, or decreases the degradation rate of autophagosome,autophagic flux assay was performed. More specifically, the conversionrate from LC3-I to LC3-II was measured in cells in the presence orabsence of NH₄Cl, which inhibits degradation of autophagosome but doesnot affect autophagosome formation, using Western blotting. The resultsare illustrated in FIG. 20.

As illustrated in FIG. 20, it is confirmed that there is no significantchange in the amount of LC3-II when treating cells with ASM and NH₄Cl.

Also, when treating with NH₄Cl or ASM after culturing fibroblast ofhuman with Alzheimer's disease in a serum-free medium or completemedium, the conversion rate from LC3-I to LC3-II was measured usingWestern blotting. The results are illustrated in FIG. 21 and FIG. 22.

As illustrated in FIG. 21, it is confirmed that the LC3-II levelincreased significantly when adding NH₄Cl after inducing autophagy byculturing cells in a serum-free medium. As illustrated in FIG. 22, it isconfirmed that the accumulation of LC3-II increased significantly whentreating with ASM after inducing autophagy by culturing cells in aserum-free medium.

Through the above test results, it is confirmed that ASM does not inducethe formation of autophagosome in Alzheimer's disease, but inhibitsproteolytic activity of autophagosome.

Example 4. Verification on Pathological Improvement Effect of ASMInhibition in Alzheimer Disease Model Mice_Confirmation of TherapeuticEffect Using ASM Inhibitory Compound, Parabiotic System and ASMDeficient Serum

4-1. Verification on Effect of Treating Alzheimer's Disease afterAdministering AMI into APP/PS1 Mice

AMI (amitriptyline-hydrochloride), which is a known inhibitor of ASMthat can cross the blood-brain barrier (BBB), was administered toAPP/PS1 mice, which are Alzheimer's disease model mice, for four months,and then the water maze (WM) test was performed (FIG. 23). The degree ofASM expression was measured by obtaining serum and brain tissue when themice became nine month old (in the same manner as Example 1-2), and thedeposition of β-amyloid in brain tissue was measured (in the same manneras Example 1-3). The test results are illustrated in FIGS. 24 to 26,respectively.

As illustrated in FIG. 24, it is confirmed that ASM decreased in theserum and brain tissue of mice where AMI was administered. Asillustrated in FIG. 25, it is confirmed that the deposition of β-amyloidwas inhibited in the cerebral cortex and hippocampus of mice where AMIis administered. As illustrated in FIG. 26, it is confirmed that theability to remember in mice where AMI is administered was recovered bydecrease in escape latency, as compared to the control group.

4-2. Verification on Effect of Treating Alzheimer's Disease UsingParabiotic System

The effect of ASM inhibition is confirmed using parabiotic system ofAPP/PS1 mice. More specifically, isochronic (APP/PS1-APP/PS1: parabioticunion between APP/PS1 mice, which are Alzheimer's disease model mice),heterochronic I (APP/PS1-ASM^(+/−): parabiotic union between APP/PS1mice and ASM^(+/−) mice), heterochronic II (APP/PS1-WT: parabiotic unionbetween APP/PS1 mice and wild mice) mice were prepared by sharing bloodflow after connecting the skin and soft tissue by surgical methods andinducing new angiogenesis between two mice (FIG. 27). The expressiondegree of ASM was measured by obtaining serum and brain tissue in thesame manner as Example 4-1, and the deposition of β-amyloid andexpression of protein in brain tissue were measured. The test resultsare illustrated in FIGS. 28 to 30, respectively.

As illustrated in FIG. 28, it is confirmed that the ASM concentration islow in the serum and brain tissue of heterochronic I (APP/PS1-ASM^(+/−))mice, as compared to isochronic (APP/PS1-APP/PS1) and heterochronic II(APP/PS1-WT) mice, and thus ASM^(+/−) mice play the role of lowering ASMconcentration.

Also, as illustrated in FIG. 29, it is confirmed that the deposition ofβ-amyloid decreased remarkably in the cerebral cortex and hippocampus ofheterochronic I (APP/PS1-ASM^(+/−)) mice, as compared to isochronic(APP/PS1-APP/PS1) mice.

Also, as illustrated in FIG. 30, it is confirmed that the conversion toLC3-II decreased, the expression of p62 and cathepsin D decreased, andthe expression of TFEB and Lamp1, which are proteins relating to ALPfunction, increased in the brain tissue of heterochronic I(APP/PS1-ASM^(+/−)) mice, as compared to isochronic (APP/PS1-APP/PS1)mice.

4-3. Verification on Effect of Treating Alzheimer's Disease Using SerumInjection

The serum of APP/PS1 or ASM^(−/−) mice was injected into APP/PS1 mice.More specifically, blood was obtained from the heart of APP/PS1 orASM^(−/−) mice, and then collected in a tube coated with EDTA. Thecollected blood was centrifuged to obtain serum, and 100 μl of serum wasintravenously injected into an eight-month old APP/PS1 mice eight timesduring 3 weeks (FIG. 31). After the test was completed, the degree ofASM expression was measured by obtaining the serum and brain tissue inthe same manner as in Example 4-1, and the deposition of β-amyloid andexpression of protein in brain tissue were measured. Also, a behaviortest was performed in the same manner as Example 1-4.

The test results are illustrated in FIGS. 32 to 36.

As illustrated in FIG. 32, it is confirmed that the ASM decreased in theserum and brain tissue of APP/PS1 mice provided with serum of ASM^(−/−)mice, as compared to the APP/PS1 mice provided with serum of APP/PS1mice.

As illustrated in FIG. 33 and FIG. 34, it is confirmed that thedeposition of β-amyloid decreased (FIG. 33) in the brain tissue ofAPP/PS1 mice provided with serum of ASM^(−/−) mice, as compared to theAPP/PS1 mice provided with serum of APP/PS1 mice. Thus, conversion toLC3-II decreased, and the expression of p62 and cathepsin D decreased(FIG. 34) in the APP/PS1 mice provided with serum of ASM^(−/−) mice, ascompared to the APP/PS1 mice provided with serum of APP/PS1 mice.

As illustrated in FIG. 35 and FIG. 36, as a result of MWM and fearconditioning test, it is confirmed that the ability to remember improvedin APP/PS1 mice provided with serum of ASM^(−/−) mice, as compared toAPP/PS1 mice provided with serum of APP/PS1 mice.

Example 5. Verification on Pathological Improvement Effect of ASMInhibition in Alzheimer Disease Model Mice_Confirmation of TherapeuticEffect Using an Antibody and an Interfering RNA to ASM ExperimentalMethod

1) Cell Culture

Human fibroblast cell line (normal cell line and PS1-overexpressing cellline) were obtained from Coriell Institute and cultured in DMEMcontaining 15% FBS under the condition of 5% CO₂ at 37° C. Thereafter,the cell lines were treated with the ASM antibody (3 μg/ml, R&D system)for 24 hours, and the ASM activity was measured.

2) Mice

The animal test conducted was approved by the Kyungpook NationalUniversity Institutional Animal Care and Use Committee (IACUC).Transgenic mouse line overexpressing APPswe (hAPP695swe) and PS1(presenilin-1M146V) based on C57BL/6 mice (Charles River, UK) were usedas Alzheimer's animal models [hereinafter, APP mice: mice overexpressingAPPswe, PS1 mice: overexpressing presenilin-1M146V; GlaxoSmithKline]. Inorder to confirm the therapeutic effect of ASM inhibition againstAlzheimer's disease using Smpd1 miR RNAi, control miR RNAi or Smpd1 miRRNAi was injected twice a week (a total 8 weeks) into theabove-mentioned mouse model (7 month-old) with a dose of 50 g/micethrough tail vein. One month after the injection of Control miR RNAi orSmpd1 miR RNAi, behavioral analysis was performed, and mouse braintissue was sampled after the behavioral analysis. This experimentaloutline is shown in FIG. 40.

3) microRNA Preparation for ASM (Smpd1 miR RNAi Construction)

ASM microRNA (Smpd1 miR RNAi), which can inhibit ASM, was constructed totest the therapeutic effect of inhibition of ASM activity in Alzheimer'sanimal model. Smpd1 miR RNAi was produced using the BLOCK-iT™ Pol II miRRNAi Expression Vector Kits (Invitrogen). First, miR RNA(Mmi520028_top_Smpd1, Mmi520028_bot_Smpd1, Mmi520030_top_Smpd1,Mmi520030_bot_Smpd1, Mmi520031_top_Smpd1 and Mmi520031_bot_Smpd1, seeTable 1) for mouse Smpd1 was cloned into pcDNA6.2-GW/miR according tothe manufacturer's protocol to generate pcDNA6.2-GW/mSmpd1 miR. Afterthat, the CMV promoter was replaced with the human Endoglin promoter 49to obtain phEndoglin mSmpd1 miR(Smpd1 miR RNAi). Control miR RNAi (seeTable 1) was also obtained in the same manner. The prepared Smpd1 miRRNAi (50 μg) was diluted in 50 μl of 10% Glucose solution and RNAse freewater was added to a final volume of 100 μl. In another tube, 6.4 μl ofin vivo-jetPEI™ (Polyplus-transfection) was diluted in 50 μl of 10%Glucose solution and RNAse free water was added to a final volume of 100μl. Then 100 μl of in vivo-jetPE™ solution was mixed with Smpd1 miR RNAisolution. After the mixed solution was reacted at room temperature for15 minutes, 200 μl of the mixed solution was injected through the tailvein of the Alzheimer's animal model twice a week.

TABLE 1 Smpd1 miRNA sequence (5′→3′) Mmi520028_top_Smp (SEQ ID NO. 1) d1TGCTGAAAGGAGTCCCACTCTGGGTGGTTTTG GCCACTGACTGACCACCCAGAGGGACTCCTTTCAGGACGACTTTCCTCAGGGTGAGACCCACCA AAACCGGAGACTGACTGGTGGGTCTCCCTGAGGAAACTCC Mmi520028_bot_Smp (SEQ ID NO. 2) d1GGAGTTTCCTCAGGGAGACCCACCAGTCAGTC TCCGGTTTTGGTGGGTCTCACCCTGAGGAAAGTCGTCCTGAAAGGAGTCCCTCTGGGTGGTCAG TCAGTGGCCAAAACCACCCAGAGTGGGACTCCTTTCAGCA Mmi520030_top_Smp (SEQ ID NO. 3) d1TGCTGATTGGTTTCCCTTTATGAAGGGTTTTG GCCACTGACTGACCCTTCATAGGGAAACCAATCAGGACGACTAACCAAAGGGAAATACTTCCCA AAACCGGTGACTGACTGGGAAGTATCCCTTTGGTTAGTCC Mmi520030_bot_Smp (SEQ ID NO. 4) d1GGACTAACCAAAGGGATACTTCCCAGTCAGTC ACCGGTTTTGGGAAGTATTTCCCTTTGGTTAGTCGTCCTGATTGGTTTCCCTATGAAGGGTCAG TCAGTGGCCAAAACCCTTCATAAAGGGAAACCAATCAGCA Mmi520031_top_Smp (SEQ ID NO. 5) d1TGCTGAACAGAGCCAGAACCAGCGCCGTTTTG GCCACTGACTGACGGCGCTGGCTGGCTCTGTTCAGGACGACTTGTCTCGGTCTTGGTCGCGGCA AAACCGGTGACTGACTGCCGCGACCGACCGAGACAAGTCC Mmi520031_bot_Smp (SEQ ID NO. 6) d1GGACTTGTCTCGGTCGGTCGCGGCAGTCAGTC ACCGGTTTTGCCGCGACCAAGACCGAGACAAGTCGTCCTGAACAGAGCCAGCCAGCGCCGTCAG TCAGTGGCCAAAACGGCGCTGGTTCTGGCTCTGTTCAGCA Control miRNA sequence miR-neg control- (SEQ ID NO. 7) topTGCTGAAATGTACTGCGCGTGGAGACGTTTTG GCCACTGACTGACGTCTCCACGCAGTACATTTCAGGACGACTTTACATGACGCGCACCTCTGCA AAACCGGTGACTGACTGCAGAGGTGCGTCATGTAAAGTCC miR-neg control- (SEQ ID NO. 8) botGGACTTTACATGACGCACCTCTGCAGTCAGTC ACCGGTTTTGCAGAGGTGCGCGTCATGTAAAGTCGTCCTGAAATGTACTGCGTGGAGACGTCAG TCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTCAGCA

4) Measurement of ASM Activity

The concentration level of ASM was measured as follows. Specifically, 3μl of mouse serum, brain tissue and fibroblast samples were mixed withASM activity buffer and stored at 37° C. 114 μl of ethanol was added toterminate the hydrolysis reaction, followed by centrifugation. 30 μl ofsupernatant was transferred to a glass vial, of which 5 μl was appliedto the UPLC system. The ASM concentration levels were quantified bycomparison with sphingomyelin and Bodipy (aminoacetaldehyde) conjugatedwith ceramide. The extraction and quantification of the sphingomyelinand ceramide was carried out by extracting and drying the lipids fromthe sample according to a known conventional method, and resuspendingthe dried lipid extract to 25 μl of 0.2% Igepal CA-630 (Sigma-Aldrich),and the concentration levels of each lipid were quantitated using theUPLC system.

5) Immunofluorescence Staining Method

After fixing the cerebral and hippocampal tissues of the mice, 0.5%thioflavin S (Sigma-Aldrich), anti-20G10 antibodies (mouse, 1:1000)against Aβ42, anti-G30 antibodies (rabbit, 1:000) against Aβ40,anti-GFAP antibodies (rabbit, 1:500, DAKO), or anti-Iba-1 antibodies(rabbit, 1:500, Wako) were co-cultured. The tissues were analyzed usinga laser scanning confocal microscope equipped with Fluoview SV1000imaging software (Olympus FV1000, Japan) or an Olympus BX51 microscope.Percentage of stained area to total tissue area was quantified andanalyzed using Metamorph software (Molecular Devices).

6) Western Blot

Western blotting method was used to analyze the expression of thefollowing genes. Western blotting was performed by a conventional methodusing an antibody against LC3 (LC3-I and LC3-II), Beclin-1, p62 [all ofthem were purchased from cell signaling Technologies], Cathepsin D (R&Dsystems), Lamp1 (abcam), TFEB (Invitrogen) and β-actin (Santa Cruz).Densitometric quantification was performed using ImageJ software (USNational Institutes of Health).

7) Real-Time Quantitative PCR

To quantify the expression levels of inflammatory cytokines (TNF-α,IL-1β and IL-6), real-time quantitative PCR was used. Total RNAs wereextracted from brain tissue using RNeasy Plus mini kit (Qiagen, Korea,Ltd.), and cDNA was synthesized from 5 μg of total RNAs using a kit ofClontech inc. (Mountain View, Calif.). Also, using Corbett researchRG-6000 real-time PCR instrument, real-time quantitative PCR wasperformed with repeated 40 cycle (one cycle consisting of 95° C., 10minutes; 95° C., 10 seconds; 58° C., 15 seconds; 72° C., 20 seconds).Table 2 shows the primers used for the real-time quantitative PCR.

TABLE 2 Sequence (5′→3′) mTNF-α (SEQ ID NO. 9) (SEQ ID NO. 10)GAT TAT GGC TCA GGG GCT CCA GTG AAT TCG TCC AA GAA AG mIL-1β(SEQ ID NO. 11) (SEQ ID NO. 12) CCC AAG CAA TAC CCA GCT TGT GCT CTG CTTAAG AA GTG AG mIL-6 (SEQ ID NO. 13) (SEQ ID NO. 14) CCG GAG AGG AGA CTTTTG CCA TTG CAC AAC CAC AG TCT TT mGAPDH (SEQ ID NO. 15) (SEQ ID NO. 16)TGA ATA CGG CTA CAG AGG CCC CTC CTG TTA CAA CA TG

8) Behavioral Experiment

MWM (Morris water maze) experiments were performed in a conventionalmanner to identify potential effects on learning and memory. Briefly,for the MWM test, mouse were learned the task four times a day for 10days, followed by the removal of the platform on the 11th day andsubsequently a probe trial performed.

9) Statistical Analysis

For the comparison of the two groups, student's t-test was performed.For comparison of multiple groups, repeated analysis of Tukey's HSD testand distribution test were conducted according to the SAS statisticalpackage (release 9.1; SAS Institute Inc., Cary, N.C.). *p<0.05,**p<0.01, ***p<0.001 were considered to be significant.

10) Acknowledgements

This research was supported by the Basic Science Research Program(2017R1A2A1A17069686, 40% contribution) of the National ResearchFoundation (NRF) of Korea funded by the Ministry of Science, ICT &Future Planning (2017R1A4A1015652, 40% contribution), Republic of Korea.This patent application was also supported by a grant of the KoreaHealth Technology R&D Project through the Korea Health IndustryDevelopment Institute (KHIDI), funded by the Ministry of Health &Welfare, Republic of Korea (HI16C2131, 20% contribution).

Experimental Results

5-1. Confirmation of ASM Activity Change after Treating ASM Antibody inFibroblasts of Patients with Alzheimer's Disease

In order to confirm the effect of ASM inhibition on Alzheimer's diseasein vitro, ASM activity was measured after fibroblasts (PS1 fibroblasts)derived from Alzheimer's patients were treated with anti-ASM antibody (3μg/ml, R&D system).

As shown in FIG. 38, ASM activity was significantly increased in the PS1fibroblasts (derived from Alzheimer's disease patients) compared withnormal human fibroblasts, but this was markedly decreased by ASMantibody treatment.

5-2. Confirmation of Effect on Autophagy-Related Protein after TreatingASM Antibody in Fibroblasts of Patients with Alzheimer's Disease

In order to identify how ASM inhibition by ASM antibody treatmentaffects autophagy-related pathways in Alzheimer's fibroblasts, theconversion of LC3-I into LC3-II and the expression levels of Beclin-1,cathepsin D, p62, Lamp1 and TFEB was confirmed by western blottingexperiments after the ASM antibody treatment, respectively.

As shown in FIG. 39A and FIG. 39B, it was confirmed that the conversionof LC3-I to LC3-II was increased in Alzheimer's fibroblasts (PS1fibroblasts) as compared with normal fibroblasts, while the conversionwas reduced by ASM antibody treatment. The expression of Beclin-1 wasnot significantly different among the three groups. And, similarly, theexpression level of p62 (an indicator of autophagy turnover), which isincreased in Alzheimer's patients, is reduced by ASM antibody treatment.In addition, the expression of Lamp1 and TFEB, an indicator of lysosomesinvolved in autophagy, was decreased in Alzheimer's fibroblasts butincreased by ASM antibody treatment. These results suggest that abnormalautophagy induced by increased ASM in Alzheimer's fibroblasts may beameliorated by ASM inhibition.

5-3. Confirmation of ASM Activity Change in Alzheimer's Animal ModelInjected with ASM microRNA

In order to verify the effect of alleviating Alzheimer's lesion byinhibition of ASM activity in vivo, ASM microRNA (Smpd1 miR RNAi)prepared by the present inventor was administered to Alzheimer's animalmodel (AD: APP/PS1 mouse) twice a week (for a total of 8 weeks) via tailvein (see FIG. 40)

First, in order to confirm whether ASM activity is inhibited or not,plasma and brain tissues of Alzheimer's animal model injected withControl miR RNAi or Smpd1 miR RNAi were collected, and ASM activity wasmeasured. As a result, it was confirmed that the level of ASMconcentration in the plasma (FIG. 41A) and the brain tissue (FIG. 41B)of the animal model of Alzheimer's injected with Smpd1 miR RNAi wasremarkably low.

5-4. Confirmation of Amyloid-β Deposition in Alzheimer's Animal ModelInjected with ASM microRNA.

To confirm whether inhibition of ASM activity by the Smpd1 miR RNAiinjection is therapeutically effective on Alzheimer's lesion or not, thecerebral cortex and hippocampus regions of the experimental group andthe control group mouse of Example 5-3 were stained with thioflavin S(ThioS), and fibrillary amyloid-β deposition was confirmed.Immunofluorescent staining of Aβ40 and Aβ42 was also performed toconfirm amyloid-β deposition.

Experimental results as shown in FIG. 42A and FIG. 42B, It was confirmedthat fibrinous Aβ deposits were less in the cerebral cortex andhippocampal region of the Alzheimer's animal model injected with Smpd1miR RNAi, compared to control miR RNAi injected Alzheimer's animalmodel.

FIG. 43A and FIG. 43B show the result of immunofluorescence staining ofAβ40 and Aβ42, respectively. It was confirmed that deposition of Aβ40(FIG. 43A) and Aβ42 (FIG. 43B) was significantly lower in the cerebralcortex and hippocampal region of the Alzheimer's animal model injectedwith Smpd1 miR RNAi than control miR RNAi injected Alzheimer's animalmodel.

5-5. Confirmation of Memory Improvement in Alzheimer's Animal ModelInjected with ASM microRNA.

To confirm whether ASM inhibition by Smpd1 miR RNAi injection inAlzheimer's animal models have a potential effect on memory ability ornot, MWM (Morris water maze) test was performed on the experimentalgroup and the control group mice of Example 5-3.

FIG. 44A, FIG. 44B, and FIG. 44C show the results of MWM test. It wasconfirmed that the recovery degree of learning and cognitive function issignificantly high in Alzheimer's animal models injected with Smpd1 miRRNAi. Specifically, an Alzheimer's animal model injected with ControlmiR RNAi showed severe impairment of spatial memory, cognition, andmemory formation, but the Alzheimer's animal model that injected Smpd1miR RNAi showed an improvement in such impairments.

5-6. Confirmation of Changes of Neuroinflammation in Alzheimer's AnimalModel Injected with ASM microRNA.

To confirm the effect of inhibition of ASM by the injection of Smpd1 miRRNAi in Alzheimer's animal model on neuroinflammatory changes, thepresent inventors observed changes in astrocyte cells (using GFAP as amarker) and microglia (using Iba-1 as a marker) in the brain of theexperimental group and the control group mouse of Example 5-3.

FIG. 45A, FIG. 45B and FIG. 45C show the results of confirming thatincreased neuroinflammation in the Alzheimer's animal model is reducedby Smpd1 miR RNAi injection, respectively. Specifically, it wasconfirmed that the activity of astrocytes and microglia was markedlylowered in the Alzheimer's animal model administered Smpd1 miR RNAi ascompared with the Alzheimer's animal model injected with Control miRRNAi (see FIG. 45A and FIG. 45B). In the Alzheimer's animal modelinjected with Control miR RNAi, the gene expression of the inflammatorycytokines TNF-□, IL-1□ and IL-6 was significantly increased comparedwith that of wild type mice. But, in Alzheimer's animal modeladministered with Smpd1 miR RNAi, the expression of the inflammatorycytokines was restored to their normal levels (FIG. 45C). These resultssuggest that inhibition of ASM activity by Smpd1 miR RNAi injectionregulates neuroinflammation in Alzheimer's brain environment.

5-7. Confirmation of Effect on Improvement of Autophagy in Alzheimer'sAnimal Model Injected with ASM microRNA.

In order to identify how ASM inhibition by Smpd1 miR RNAi administrationaffects autophagy-related pathways, the conversion of LC3-I into LC3-IIand the expression levels of Beclin-1, cathepsin D, p62, Lamp1 and TFEBwas confirmed in brain tissue samples of the experimental group andcontrol group mice of Example 5-3 by western blotting experiments.

As shown in FIG. 46A and FIG. 46B, it was confirmed that the conversionof LC3-I to LC3-II was increased in an Alzheimer's animal model (APP/PS1mice) in which Control miR RNAi was administered, compared withwild-type (WT) mice. The conversion was reduced in APP/PS1 mice treatedwith Smpd1 miR RNAi. The expression of Beclin-1 was not significantlydifferent among the three groups. And, the expression level of p62 (anindicator of autophagy turnover), which is increased in Alzheimer'spatients, is reduced by injection of Smpd1 miR RNAi. In addition, theexpression of Lamp1 and TFEB, an indicator of lysosomes involved inautophagy, was decreased in Alzheimer's animal model administered withControl miR RNAi, but increased by injection of Smpd1 miR RNAi. Theseresults demonstrate that abnormal autophagy induced by increased ASM inthe brains of Alzheimer's animal models can be improved by ASMinhibition by Smpd1 miR RNAi injection

Taking all the above results into consideration, it was demonstratedthat inhibition of ASM activity by ASM antibody treatment in Alzheimer'sfibroblasts could regulate abnormally damaged autophagy. In addition, Itwas demonstrated that inhibition of ASM activity by injection of Smpd1miR RNAi in Alzheimer's animal model could reduce Aβ plaque depositionand inflammation, and restore damaged autophagy, and improve learningand memory ability. Therefore, it has been found that ASM activity andexpression inhibitors such as ASM antibody and ASM microRNA can be usedas a preventive or therapeutic agent for neurodegenerative diseases suchas Alzheimer's disease.

Example 6. Activity of ASM microRNA in Improving Depression in AnimalModels 6-1. Preparation of Test Animals

For depression-induced animal model, C57BL/6 mice (Charles River, UK)were used as those with depression by inducing Repeated Social Defeat(RSD) stress for 10 days. Repeated Social Defeat (RSD) stress wasinduced by placing two male, 6-8 week-old C57BL/6 mice with one male,6-8 week-old CD-1 mouse (aggressive intruder mouse) in the same cage for10 days, 2 hours daily. After 10 days, depression of C57BL/6 mice wasconfirmed through the depression behavior test.

To confirm the therapeutic effect of ASM inhibitor (Smpd1 miR RNAi) inthe depressed animal model, 6-week-old male C57BL/6 mice were injectedwith said ASM inhibitor via tail vein injection twice weekly in a dosageof 50 μg/mice for 4 weeks prior to the induction of depression. Fourweeks later, behavioral analysis was performed after inducing RepeatedSocial Defeat (RSD) stress for 10 days, followed by obtaining plasmasamples of the mice (FIG. 47A).

6-2. Changes in ASM Activity in Depression-Induced Animal ModelsAdministered with ASM microRNA

For verifying the effect of alleviating depression lesions in vivo byinhibiting ASM activity, ASM microRNAs (Smpd1 miR RNAi) prepared by theinventors were administered intravenously twice a week to the prepareddepression-induced animal models as describe above (RSD stress inducedmice; WT/RSD) (FIG. 47A).

In order to confirm whether the ASM activity is inhibited, plasma of adepression-induced animal model injected with Control miR RNAi or Smpd1miR RNAi was extracted to determine ASM activity. As a result, it wasconfirmed that the level of ASM concentration in the plasma of thedepression-derived animal model injected with Smpd1 miR RNAi (FIG. 47B)was significantly low.

6-3. Improvement of Depression in Depression-Induced Animal ModelsAdministered with ASM microRNA

To determine whether ASM inhibition by Smpd1 miR RNAi injection leads tothe improvement of depression in a depression-induced animal model, anOpen field test, a Dark & Light test, a Tail suspension test and Forceswim test were performed, respectively.

As shown in FIG. 48A, FIG. 48B, FIG. 48C, and FIG. 48D, thedepression-induced animal model injected with Control miR RNAi showedsevere symptoms of depression, whereas the depression animal modeladministered with Smpd1 miR RNAi was found to significantly improve thesymptoms of depression.

Based on the above results, it can be seen that inhibition of ASMactivity by Smpd1 miR RNAi injection in depression induced animal modelcan improve the conditions or symptoms of depression. Therefore, it issuggested that ASM expression inhibitors such as ASM microRNA can beused as a preventive or therapeutic agent for degenerative neurologicaldisorders including depression.

Hereinafter, preparation examples of the pharmaceutical composition andfood composition of the present invention are described for illustrativepurposes only, and the present invention is not intended to be limitedby the following preparation examples.

Preparation Example 1. Preparation of a Pharmaceutical Formulation 1-1.Preparation of Powders

-   -   ASM expression inhibitor or activity inhibitor 2 g    -   lactose 1 g

The above ingredients were mixed and filled into a sealed pouch toprepare a powder formulation.

1-2. Preparation of a Tablet

-   -   ASM expression inhibitor or activity inhibitor 100 mg    -   corn starch 100 mg    -   lactose 100 mg    -   stearic acid magnesium 2 mg

The above ingredients were mixed, and then tabulated according to aconventional tablet preparation method to prepare a table formulation.

1-3. Preparation of a Capsule

-   -   ASM expression inhibitor or activity inhibitor 100 mg    -   Corn starch 100 mg    -   lactose 100 mg    -   stearic acid magnesium 2 mg

The above ingredients were mixed, and then filled into a gelatin capsuleaccording to a conventional capsule preparation method to provide acapsule formulation.

Preparation Example 2. Preparation of Food Formulation 2-1. Preparationof Health Care Food

ASM expression inhibitor or activity inhibitor 100 mg vitamin mixtureproper quantity vitamin A acetate 70 g vitamin E 1.0 mg vitamin B1 0.13mg vitamin B2 0.15 mg vitamin B6 0.5 mg vitamin B12 0.2 g vitamin C 10mg biotin 10 g nicotinic acid amid 1.7 mg folic acid 50 g calciumpantothenate 0.5 mg inorganic mixture proper quantity ferrous sulfate1.75 mg zinc oxide 0.82 mg magnesium carbonate 25.3 mg potassiumphosphate monobasic 15 mg calcium phosphate dibasic 55 mg potassiumcitrate 90 mg calcium carbonate 100 mg magnesium chloride 24.8 mg

In the above composition ratio including vitamins and minerals, theingredients are mixed in a ratio appropriate for a health care food, butthe mixing ratio may be changed. A health care food composition may beprepared according to a conventional method of preparing a health carefood, the method including the steps of mixing the above ingredients,preparing granules, and using the granules in the same manner as theconventional method.

What is claimed is:
 1. A method for treating depression in a subject inneed thereof, comprising administering to a subject a therapeuticallyeffective amount of a composition comprising an acid sphingomyelinase(ASM) expression inhibitor as an active ingredient, wherein the ASMexpression inhibitor comprises a miRNA which complementarily binds tomRNA of ASM gene and comprises at least a nucleic acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO:5.